We seek to understand the detailed mechanisms for the generation of the endocochlear potential (EP), an extracellular positive potential (~80 mV) that boosts the driving force for the influx of cations into hair cells during mechanoelectrical transduction. The importance of EP is underpinned by the fact that drugs whose effects decrease EP are ototoxic and experimental manipulations that abolish EP result in a decreased hearing threshold or total deafness. We hypothesize that the EP is produced and maintained by a cadre of K+ channels in the apical membrane of intermediate cells (ICs) and marginal cells (MCs), as well as basolateral Cl- channels in conjunction with NKCC1 and Na+/K+ATPase. We further predict that K+ regulation in the cochlear duct is tightly linked to the activity of K+ channels in cells of the medial wall (Reissner's membrane, RM) and endolymphatic sac (ES). We have made substantial progress towards the objectives of the proposal in the last grant cycle. For the next grant cycle, we will focus our attention on: 1) Clarifying unresolved aspects of the identity, and elementary properties of the subtypes of K+ channels, in cells of medial and lateral walls (MWs & LWs) of the cochlear duct (CD). We will extend these fundamentally important studies to the endolymphatic sac and duct (ES/D). 2) Determining the molecular identity, cellular localization, and density of cell-specific K+ channels in cells of cochlear MW, LW and ES/D. 3) Identifying distinct features of K+ and Cl- channels, binding partners of the channels and their stoichiometry, their density, and polarity of expression that endow their unequaled traits in the CD to confer EP. 4) Exploiting important features of the embryology of cells of the CD and cell-specific expression of genes/promoters to generate mouse models with cell-specific deletions/alterations of K+ channels. This will test the hypothesis that K+ regulation, EP generation, and maintenance in the inner ear is dependent on cell-specific expression of K+ channels in the MW and LW of the CD. We will deploy innovative molecular biological, electrophysiological, and imaging techniques, many inspired from previous cochlear duct K+ channel studies, to the discovery of fundamental, newly accessible arenas of K+ channel physiology and the mechanisms for the generation of the EP and K+ homeostasis in the inner ear. Collectively, these studies will substantially expand our understanding of the cellular mechanisms for the generation of EP. Of pragmatic importance in these studies is the tantalizing possibility of developing strategies that may be used to alleviate hearing loss associated with K+ channel malfunction in the inner ear.